Cancer is the second most common disease and also one of the most feared. Cancer occurs when cells continue to divide and fail to die at the appropriate time. Under normal circumstances, the many types of cells that make up the body grow and divide to produce more cells as they are needed in order to maintain a healthy body. Tumors may form when this orderly process is disrupted by changes in genes that control normal cell growth and death and cellular growth becomes uncontrolled. Genetic changes that arise internally due to defective DNA repair or may be induced by external factors such as diet, exposure to ultraviolet or other types of ionizing radiation, viruses such as cervical papillomaviruses, exposure to chemical carcinogens in the workplace or in the environment, drug or tobacco use, or to agents such as asbestos. Some detrimental genetic alterations are inherited.
During the transformation process, malignant neoplasms grow into a disorganized mass, however, they usually retain some resemblance to the normal tissue from which they arise. Upon histological examination, tumors can be classified according to cell type origin. For example, tumors of epithelial origin are classed as carcinomas. Sarcomas arise from tissues of mesodermal origin. Carcinomas and sarcomas can be further distinguished as adenocarcinomas, hepatocarcinomas, osteosarcomas or fibrosarcomas. Other types of cancers include leukemias and various types of tumors of primitive origin such as neuroblastoma and meduloblastoma. Malignant cancers can affect humans as well as many animal species.
It is well understood that the main reason all cancer cells are not removed from the body is because these cells are seen by the immune system as “self”; i.e., they are the host's own cells, and because they are poorly immunogenic.
In order to develop immunotherapies for the treatment of cancer, the different ways parasitic tumor cells evade the immune system are taken into consideration, particularly in how these abnormal cells develop. Although each tumor is thought to begin by the clonal reproduction of a single cell, additional changes eventually give rise to a heterogeneous mixture of different subclones, which are in effect antigenic variants. Once under the selective pressure of the host's immune response, low antigenic variants gain advantage over subclones that express fewer or more immunogenic molecules. The less immunogenic and the lower the density of the tumor-associated antigens on the plasma membrane, the more likely the tumor cells will fall below the threshold of immune detection and become invisible to host surveillance.
Tumor antigens are subject to antigenic modulation, i.e., the tumor antigens appear to be temporarily lost after exposure to specific antibodies, although alternatively, tumors may simply suppress the activities of immune effector cells such as T-cells and macrophages. On the other hand, a few isolated tumor cells may contain too few antigens to stimulate an effective immune response so that by the time immunity has developed, the tumor is beyond the capability of the immune system to destroy it. Some tumors may even interfere with normal immune responses by invading lymphoid tissues or secreting immunosuppressive factors.
While tumor-specific protein or peptide vaccines are by definition specific for a particular tumor, a major concern in their use is tumor heterogeneity. Although tumor cell clones expressing the tumor-specific peptide epitopes may be destroyed, clones that do not express the epitope escape immune attack, due to the fact that tumors are not clonal but are comprised of a diversity of cells.
Conventional cancer treatments typically include some form of chemotherapy involving use of drugs that are cytotoxic to the cancer cells, but also tend to kill non-cancerous cells. One approach to lowering therapeutic drug toxicity is transfection of healthy, normal stem cells with transgenes that confer resistance to these agents. In theory, this results in cytotoxic drug-resistant cells and allows the administration of higher, therapeutically significant doses of chemotherapeutic agents. Use of transfected cells has been suggested for protection of bone marrow cells since bone marrow cells are rapidly dividing and thereby most at risk to chemotoxicity and in fact has shown some success in animal models, Licht et al., 2000.
However, use of gene therapy to modify normal cells appropriate for cancer treatment has several drawbacks although in vivo treatments for malignant melanoma in dogs, for example, has met with some success. A positive response to tumor regression was observed over a period of 6-12 weeks after a direct DNA injection encoding a Staphylococcus antigen and GM-CSF cytokine (W096/36366). Liposome/Staphylococcal antigen injections alone, however, failed to show any effect even after 17 weeks, suggesting that tumor regression was caused by a toxic effect generated by the cytokine or cytokine/antigen combination in the cancer cells.
Immunotherapy methods based on manipulation of the host immune system to identify cancer cells as non-self; i.e., methods to mobilize and strengthen the immune system so that it can selectively destroy and/or inhibit proliferation of cancerous cells, is gaining more attention. This is due to the recognition that the host itself may be able to generate the safest and most effective defense against cancer.
The vast majority of malignancies arise in immunocompetent hosts, raising doubts as to whether a general strengthening of the immune system can ever be effective in targeting cancer cells, which are not always recognized as foreign by the host. Tumor cells carry tumor-associated or tumor-specific antigens that are different from their normal counterparts. Tumor-associated antigens such as oncofetal antigens are normally synthesized during embryogenesis but are not found on adult cells, can be generated by the activation of normally repressed genes. Some antigens are present but masked; while others may be lost when the cells become transformed and thus alter the profile of adjacent molecules by their absence. Antigens may also be modifications of normal molecules or may be nuclear or cytoplasmic and thus hidden from immune surveillance. Tumor-specific antigens are restricted to tumor tissues. They are not found in normal adult or fetal tissues and are rare.
Antigens, bacterial and viral, have been used in combination with cytokine or other immunomodulator genes delivered by means of adenovirus, retrovirus or plasmid vectors (WO 94/21808; WO 96/29093). The presence of cytokines may contribute to limited success of some of these approaches. In certain cases, a highly destructive and specific response to otherwise nonimmunogenic tumors can be elicited by the insertion of genes encoding interleukin-2, interleukin-4, interleukin-12, interferon-γ, interferon-α and/or tumor necrosis factor into the tumor cells as well as into cytotoxic lymphocytes or macrophages, although serious side-effects may occur at high doses.
Oncophages have been used to lyse autologous tumor cells in the hope of generating a tumor-specific response. Others have transfected tumor cells with immunotoxins (Wallack, et al., 1995). Patients also have been vaccinated with specific tumor antigens, tumor-specific monoclonal antibodies, HSP 70 purified from autologous tumor cells, autologous T cells activated against tumor cells ex vivo. These methods focus on specific aspects of the immune response to particular tumor characteristics.
Autologous tumor-infiltrating lymphocytes have been used in genetic immunomodulation studies because of their inherent specificity for the tumor and their ability to home back to the tumor site when reinfused into the patient. Normal tissue has been protected by stably transfecting normal bone marrow cells with cytokine genes prior to chemotherapy, thereby achieving a more continuous effect while obviating the need to infuse drugs which have short half-lives and produce systemic side effects when delivered intravenously (Yamaguchi, et al., 2003).
An immunostimulating vaccine has been described in U.S. patent Ser. No. 10/964,471 where autologous tumor cells were engineered to express a priming antigen, Emm55 and used to formulate a vaccine. Extensive in vivo tests in a murine model demonstrated protection from a highly invasive neuroblastoma tumor and an inhibitory/therapeutic effect when administered after tumors had developed.
There are currently just over 100 cancer vaccines in the developmental pipeline for use in humans. Collectively, they employ a diverse array of technology platforms with approximately 66% being antigen-specific, 21% being polyvalent and 14% being dendritic cell vaccines. Despite the intense interest in antigen-specific vaccines, the cell-based therapies have demonstrated the most compelling clinical data. In addition to the obvious human medical markets, there is an analogous and equally expanding veterinary cancer market for companion animals.
Both pharmaceutical and biotechnology companies are turning to the companion animal healthcare market which is currently valued in the billions of dollars and is growing at a rate of 10% per year. This market growth is in response to pet owners who are demanding better care and access to cutting-edge technology for their animals. Although the pet population has increased somewhat, the key driver for growth is the willingness of pet owners to spend and the ability of veterinarians to meet the demand. In the US alone, pet owners spend over $19 B a year on veterinary care, which is increasingly shifting to veterinary specialists including oncologists, ophthalmologists and orthopediatricians, and other specialists. For many companion animal cancers, systemic chemotherapy is the current treatment of choice even though recurrence and multi-drug resistance are common. In addition, chemotherapy is administered only as a palliative therapy, to improve and prolong life, and the pet owner is often reluctant to treat with chemotherapy because these toxic chemical commonly cause side effects such as anorexia, vomiting, diarrhea, sepsis and even death and can cost up to $5,000 over a 6 month period.
In the veterinary market, it has been estimated that 45% of dogs 10 years or older will die of cancer, and this number is increasing at a rate of 38% per year in some states. While lymphoma is not breed-specific, an example of the incidence of lymphoma in Golden Retrievers (60,000 per year, which is 1 in 8) provides an indication of the nature of the market for this type of cancer. Each year, there are approximately 10,000 new cases of osteosarcoma in dogs and cancer accounts for 60% of all Golden Retriever deaths. The total canine cancer market in the US alone can be conservatively calculated by assuming a 0.3% incidence of cancer in the US population of 64,000,000 pet dogs. Estimates for the US canine oncology market are approximately $192 M. Estimates for the development of animal immunotherapeutic treatments for all companion animals, including cats, horses and birds as well as dogs, represents a market opportunity for annual revenues of $3 B.